Site-Specific Albumin-Selective Ligation to Human Serum Albumin under Physiological Conditions

Human serum albumin (HSA) is the most abundant protein in human blood plasma. It plays a critical role in the native transportation of numerous drugs, metabolites, nutrients, and small molecules. HSA has been successfully used clinically as a noncovalent carrier for insulin (e.g., Levemir), GLP-1 (e.g., Liraglutide), and paclitaxel (e.g., Abraxane). Site-specific bioconjugation strategies for HSA only would greatly expand its role as the biocompatible, non-toxic platform for theranostics purposes. Using the enabling one-bead one-compound (OBOC) technology, we generated combinatorial peptide libraries containing myristic acid, a well-known binder to HSA at Sudlow I and II binding pockets, and an acrylamide. We then used HSA as a probe to screen the OBOC myristylated peptide libraries for reactive affinity elements (RAEs) that can specifically and covalently ligate to the lysine residue at the proximity of these pockets. Several RAEs have been identified and confirmed to be able to conjugate to HSA covalently. The conjugation can occur at physiological pH and proceed with a high yield within 1 h at room temperature. Tryptic peptide profiling of derivatized HSA has revealed two lysine residues (K225 and K414) as the conjugation sites, which is much more specific than the conventional lysine labeling strategy with N-hydroxysuccinimide ester. The RAE-driven site-specific ligation to HSA was found to occur even in the presence of other prevalent blood proteins such as immunoglobulin or whole serum. Furthermore, these RAEs are orthogonal to the maleimide-based conjugation strategy for Cys34 of HSA. Together, these attributes make the RAEs the promising leads to further develop in vitro and in vivo HSA bioconjugation strategies for numerous biomedical applications.


S2.1. Library Design
The library was composed of 35 natural and unnatural amino acids summarized in Table S1 Table S1. Amino Acid Used in Library Fmoc-Aib-OH

S2.2 Library Synthesis
Figure S1 Tri-peptide KX2X1 based peptidomimetic OBOC Library Split-mix strategy was used in library synthesis to yield tri-, tetra-and pentapeptide based peptidomimetic libraries shown in Figures S1-S3, respectively. 2.0 g of TentaGel beads (NH2 loading: 0.31 mmol/g) was swollen in DMF overnight before library synthesis. In brief, a library synthesis cycle starts with splitting the beads into 42 5mL plastic tubes equally. To each tube was added 1 mL of 0.2 M corresponding amino acid solution, 1 mL of 0.2 M 6-Cl HOBt solution, and 50 µL of DIC. The coupling took 2 hours, and the completion of coupling was monitored by the ninhydrin test. After the coupling was done, the beads were combined in a 10 mL disposable polypropylene column with a polyethylene frit and the beads were washed with DMF, methanol and DMF, three times for each, before the addition of 20% 4-methyl piperidine in DMF to remove Fmoc protecting group (5 min, 15 min) and expose the N-terminal amine before next cycle began.
Before coupling myristic acid to the N-terminal of the peptides on beads, bi-layer beads were prepared using the bi-phasic solvent approach 1 to achieve 20% binding peptides displayed on the bead outer layer and 80% coding tag reside inside the beads. In brief, the library beads were dried completely and then swollen in water overnight. After water was drained, 178 mg Fmoc-OSu in 40 mL dichloromethane (DCM)/diethyl ether solution (v/v 55:45) was added to the beads, followed by addition of 86 µL DIEA. The column was vigorously shaken for 1 hour. The solution was drained, and the library beads were washed extensively by DMF and DCM. 432 mg Boc-anhydride in DCM was added to protect the N-terminal amino groups of coding peptides. After the Boc protection was done and confirmed by ninhydrin test, 20% 4-methyl piperidine in DMF was added to remove the Fmoc group, followed by coupling with myristic acid using 6-Cl HOBt/DIC. The Dde protecting group on lysine side chain in the library was then removed by 3% hydrazine monohydrate solution in DMF (5 min, 10 min). After extensive washing, 105 mg of acrylic acid (5 equiv.), 262 mg of 6-Cl HOBt (5 equiv.) and 239 µL of DIC (5 equiv.) was added to the beads. The coupling reached the completion in 1 hour.
The library beads were washed by DMF, MeOH, (DCM), three times each and dried under vacuum. Global deprotection was achieved with TFA cocktail (v/v, TFA: thioanisole: water: triisopropylsilane = 87.5%: 5% : 5% : 2.5%) for 3 hours. Then the TFA cocktail was drained, and the library beads were washed sequentially with DMF, DCM, MeOH, DMF, 50% DMF, water, ethanol, three time each, and stored in 70% ethanol for future screening. Figure S4 Schematic Illustration for Enzymatic Approach to Find RAE that Can Crosslink HSA The screening for peptides that covalently target specific domain of HSA is performed in double-incubation approach: incubation with antibody solution to remove the false positive beads before incubation with HSA.

S3. Enzyme-linked Immunocolorimetric Screening Procedures
In a separatory column, approximately 100mg OBOC library (~300,000 beads) was washed by water for 3 times, followed by PBS-Tween 20-Gelatin buffer (PBSTG, made by 0.05% Tween 20 (v/v), 1% gelatin (w/v), pH = 7.4) for 3 times. The library was blocked by PBSTG for 1 hour at room temperature. Then the library was incubated with 2 mL 0.5 µg/mL Anti-HSA-Alkaline Phosphatase conjugate (Anti-HSA-AP) in PBSTG buffer for another 1 hour at room temperature. The antibody solution was drained, and the OBOC library beads were washed by TBS buffer twice. 1.65 mg BCIP substrate was dissolved in 10mL TBS buffer (pH = 8.8), and 1 mL of resulting substrate buffer was added to OBOC library beads. After 1 hour, beads with blue color were picked and discarded, and the rest beads were washed by 8 M guanidine HCl for 3 times, followed by washing with alternating DMF and methanol. Finally, the library beads were washed by PBSTG for 3 times and blocked by PBSTG buffer for another 1 hour. After that the library beads received 1 mL of 7.5 nM HSA solution in PBSTG buffer and the incubation last for 1 hour. Then the beads were washed by PBS for 3 times, followed by 3 times of 100 mM glycine solution (pH = 3.0) to elute non-covalently bound HSA. The antibody incubation and color development process were repeated as described above. Beads that bear blue color were picked and sequenced by Edman-degradation microsequencer.

S4.1. Synthesis of Biotinylated Reactive Affinity Elements
Scheme S1 Synthetic flow-chart for Biotinylated HSA Reactive Affinity Elements Unless specified otherwise, the backbone of myristylated peptidomimetics was synthesized by microwave automatic peptide synthesizer (CEM) on Rinker amide MBHA resin (NH2 loading: 0.51mmol/g) using 6 equivalents DIC/Oxyma and 6 equivalents of Fmoc-protected amino acids. Each coupling cycle took 2 minutes.
To prepare biotinylated reactive affinity elements and biotinylated non-covalent peptides used in binding affinity study, Fmoc-Lys (Biotin) was coupled to Rinker amide MBHA resin through 4 equivalents of HATU, Fmoc-Lys (Biotin)-COOH and DIEA in NMP solution at room temperature for overnight. The completion of coupling was monitored by ninhydrin test. After constructing the backbone of affinity elements using a peptide synthesizer (CEM Liberty Blue 2.0), myristoyl tail was installed on N-terminal of affinity elements by 5 equivalents of myristic acid, 6-Cl HOBt and DIC for overnight at room temperature. To couple acrylic acid to lysine side chain, after the removal of Dde protecting group by 3% Hydrazine monohydrate, 5 equivalents of acrylic acid, 6-Cl HOBt and DIC were pre-mixed for half an hour before added to the resin. The coupling lasts for 1 hour at room temperature and the completion of coupling was monitored by ninhydrin tests.
To cleave the peptides off the beads, prior to cleavage the beads were washed with DMF, MeOH and DCM and thoroughly dried on vacuum. TFA cocktail (v/v, 95% TFA, 2.5% Water, 2.5% TIS) was prepared and added immediately to the dried beads and the mixture was shaken at a shaking bed for 2 hours. The TFA cocktail solution was then collected and condensed under nitrogen gas. Cold diethyl ether was added to precipitate the peptides and the resulting emulsion was enriched by centrifugation (3000 x g, 5 minutes). The ether was then discarded, and the peptides sediments were dissolved by acetonitrile/water mixture (50/50, v/v), followed by purification with Shimadzu LC-20AR Prominence liquid chromatograph suite coupled with a C18 column, starting at the mobile phase composed by ACN/Water = 70:30 containing 0.1% (v/v) TFA. The fractions containing desired products were collected or combined. The solvent was removed by lyophilization.

S4.2. Synthesis of FC-LYL1
The FC-LYL1 was synthesized from intermediate LYL1-K(NH2) that is prepared by similar solid phase peptide synthesis (SPPS) strategy described in S4.1.  Biotinylated Non-covalent RAEs was synthesized by replacing acrylamide with acetyl group to study the intrinsic binding affinity of peptidomimetics using bio-layer interferometry (BLI) assay. For BLI assay, 1 μM non-covalent biotinylated RAEs ( Figure   S6) solution was prepared by kinetic buffer as the bait. As the fish, HSA solution at various concentration prepared by serial dilution using kinetic buffer, and the last well of dilution was filled with kinetic buffer as the reference well. Using 96-well plate, the biosensors were equilibrated in kinetic buffer for 60s.

NCB-LYL4
NCB-LYLG wells, kinetic buffer wells, HSA well, kinetic buffer wells again for 120s, 60s, 480s, 960s as loading, baseline, association, and dissociation step, respectively. The data was subtracted by reference wells and processed by ForteBio Data Analysis software to derive the Kd value using steady-state approximation by 1:1 model ( Figure  2), and the condensation process was repeated 2 times to further remove biotinylated peptidomimetics. After that, the condensed HSA conjugate (typically 20-50 μL) was diluted by 100 μL PBS for Neutravidin pull-down assay.

Down Assay
Using neutravidin-immobilized agarose, the NeutrAvidin pull-down assay intercepted biotinylated proteins through biotin-avidin interaction. Therefore, the conjugation yield can be determined by measuring the difference caused by NeutrAvidin pull-down.

Scheme S4 Schematic Demonstration for Neutravidin Pull-Assay
The yield of conjugation is calculated based on the concentration difference of HSA before and after passing through Neutrvidin agarose, which is calculated by following equation (1): (1) To perform the pull-down assay, 100 μL slurry of high capacity NeutrAvidin agarose (ThermoFisher Scientific) was loaded into a separatory tube.       for desating by LC-MS water and acetonitrile.

S7.2 Database Searching Parameters
Charge state deconvolution and deisotoping were not performed. All MS/MS samples were analyzed using X! Tandem (The GPM, thegpm.org; version X! Tandem Alanine

S9.2. General Procedures for Electrophoresis & Western Blot
Samples for electrophoresis were prepared by 4X LDS sampling buffer, which was premixed with 20% 2-mercapthanol.